Buffy coat tube and float system and method

ABSTRACT

A tube and float system for use in separation and axial expansion of the buffy coat is provided. The system includes a transparent, or semi-transparent, flexible sample tube and a rigid separator float having a specific gravity intermediate that of red blood cells and plasma. The sample tube has an elongated sidewall having a first cross-sectional inner diameter. The float consists of a main body portion and one or more support members protruding from the main body portion to engage and support the sidewall of the sample tube. The main body portion and the support members of the float have a cross-sectional diameter less than that of the first cross-sectional inner diameter of the tube when the sample tube is expanded, such as by centrifugation. The main body portion of the float together with an axially aligned portion of the sidewall define an annular volume therebetween. The support members protruding from the main body portion of the float traverse said annular volume to produce one or more analysis areas. During centrifugation, the centrifugal force enlarges the diameter of the tube to permit density-based axial movement of the float in the tube. Thereafter, the centrifugal force is reduced to cause the tube sidewall to return to its first diameter, thereby capturing the float and trapping the buffy coat constituents in the analysis area. The buffy coat constituents can then be evaluated or measured.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/370,635, filed Mar. 7, 2006, now U.S. Pat. No. 7,329,534, which wasitself a divisional of U.S. patent application Ser. No. 10/263,975,filed Oct. 3, 2002, now U.S. Pat. No. 7,074,577. Both applications arehereby fully incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to density-based fluidseparation and, in particular, to an improved sample tube and floatdesign for the separation and axial expansion of constituent fluidcomponents layered by centrifugation, and a method employing the same.The present invention finds particular application in blood separationand axial expansion of the buffy coat layers, and will be described withparticular reference thereto. However, it will be recognized that thepresent invention is also amenable to other like applications.

BACKGROUND OF THE INVENTION

Quantitative Buffy Coat (QBC) analysis is routinely performed inclinical laboratories for the evaluation of whole blood. The buffy coatis a series of thin, light-colored layers of white cells that formbetween the layer of red cells and the plasma when unclotted blood iscentrifuged or allowed to stand.

QBC analysis techniques generally employ centrifugation of smallcapillary tubes containing anticoagulated whole blood, to separate theblood into essentially six layers: (1) packed red cells, (2)reticulocytes, (3) granulocytes, (4) lymphocytes/monocytes, (5)platelets, and (6) plasma. The buffy coat consists of the layers, fromtop to bottom, of platelets, lymphocytes and granulocytes andreticulocytes.

Based on examination of the capillary tube, the length or height of eachlayer is determined during the QBC analysis and converted into a cellcount, thus allowing quantitative measurement of each layer. The lengthor height of each layer can be measured with a manual reading device,i.e., a magnification eyepiece and a manual pointing device, orphotometrically by an automated optical scanning device that finds thelayers by measuring light transmittance and fluorescence along thelength of the tube. A series of commonly used QBC instruments aremanufactured by Becton-Dickinson and Company of Franklin, Lakes, N.J.

Since the buffy coat layers are very thin, the buffy coat is oftenexpanded in the capillary tube for more accurate visual or opticalmeasurement by placing a plastic cylinder, or float, into the tube. Thefloat has a density less than that of red blood cells (approximately1.090 g/ml) and greater than that of plasma (approximately 1.028 g/ml)and occupies nearly all of the cross-sectional area of the tube. Thevolume-occupying float, therefore, generally rests on the packed redblood cell layer and expands the axial length of the buffy coat layersin the tube for easier and more accurate measurement.

There exists a need in the art for an improved sample tube and floatsystem and method for separating blood and/or identifying circulatingcancer and/or other rare cells, organisms or particulates or objects(i.e., stem cells, cell fragments, virally-infected cells, trypanosomes,etc.) in the buffy coat or other layers in a blood sample. However, thenumber of cells expected to be typically present in the buffy coat isvery low relative to the volume of blood, for example, in the range ofabout 1-100 cells per millimeter of blood, thus making the measurementdifficult, particularly with the very small sample sizes employed withthe conventional QBC capillary tubes and floats.

The present invention contemplates a new and improved blood separationassembly and method that overcome the above-referenced problems andothers.

SUMMARY OF THE INVENTION

In a first aspect of the invention, a method of separating and axiallyexpanding the buffy coat constituents in a blood sample includesintroducing the blood sample into a flexible sample tube having anelongate sidewall of a first cross-sectional inner diameter. An elongaterigid volume-occupying float is also inserted into, or is present in,the flexible sample tube.

The float has a specific gravity intermediate that of red blood cellsand plasma. It includes a main body portion and one or more supportmembers protruding from the main body portion of the float to engage andsupport the sidewall of the sample tube. The main body portion and thesupport members have a cross-sectional diameter less than the firstinner diameter of the tube when the sample tube is subsequentlyexpanded, such as by centrifugation.

The main body portion of the float, together with an axially alignedportion of the sidewall of the sample tube, defines an annular volumetherebetween. The support members protruding from the main body portionof the float traverse the annular volume to engage and support thesidewall of the tube thereby producing one or more analysis areas.

The sample tube containing the blood sample and float is thencentrifuged to effect a density-based separation of the blood sampleinto discrete layers at a rotational speed that causes a resilientexpansion or enlargement of the diameter of the sidewall to a seconddiameter in response to pressure in the blood caused by the centrifugalforce, which diameter expansion is sufficiently large to permit axialmovement of the float in the tube. During centrifugation, the float ismoved into axial alignment with at least the buffy coat layers of theblood sample due to the density of the float. After centrifugation, therotational speed is reduced and the tube sidewall returns to essentiallyits first diameter and engages the float. As a result, the buffy coatconstituents are trapped in the analysis areas for review, measurementand/or detection by conventional methods.

In a further aspect of the invention, an apparatus for separation andanalysis of a target analyte in a sample of anticoagulated whole bloodis produced. The apparatus includes a transparent, or semi-transparent,flexible tube for holding the sample, the tube having an elongatesidewall of a first cross-sectional inner diameter. The apparatusfurther includes an elongate, rigid, volume-occupying float having aspecific gravity which is intermediate that of red blood cells andplasma.

The float includes a main body portion having one or more supportmembers protruding from the main body portion. The cross-sectionaldiameter of the main body portion and/or the support members of thefloat are less than the first cross-sectional inner diameter of the tubewhen the sample tube is subsequently expanded. In this regard, thesidewall is resiliently radially expandable to a second diameter inresponse to pressure or force. The second diameter is sufficiently largeto permit axial movement of the float in the tube during centrifugation.

The main body portion of the float, together with an axially alignedportion of the sidewall, defines an annular volume therebetween. Theprotrusions of the float traverse the annular volume and engage andsupport the sidewall, forming the analysis area subsequent tocentrifugation.

In another aspect, a volume occupying separator float adapted for usewith an associated sample tube is provided. The float includes a rigidmain body portion and one or more support members protruding from themain body portion of the float to engage and support the sidewall of thesample tube. The main body portion and the support members have across-sectional diameter less than an inner diameter of the sample tubewhen the sample tube is expanded. The main body portion together with anaxially aligned portion of the sidewall, define an annular volumetherebetween. Additionally, the supporting members protruding from themain body portion of the float traverse the annular volume to engage andsupport the sidewalls and to produce one or more areas for analysis.

In a still further aspect, a method for detecting circulating targetcells, such as epithelial cancer cells, stem cells, cell fragments,virally-infected cells, trypanosomes, etc., in an anticoagulated wholeblood sample is provided. This method includes combining the bloodsample with one or more target cell epitope-specific-labeling agents soas to differentiate the target cells from other cells in the bloodsample. The blood sample and a volume-occupying separator float areplaced into a transparent, or semi-transparent, flexible sample tube.The separator float has a specifically defined specific gravity. Itcomprises a rigid main body portion and tube support members. Theseparator float in conjunction with the sidewalls produces one or moreareas of analysis. Additionally, the float has a cross-sectionaldiameter less than an inner diameter of the sample tube when the sampletube is expanded. The blood sample and separator float are centrifugedin the sample tube to effect centrifugally motivated localization of anytarget cells present in the blood sample to the areas of analysis. Theblood sample present in the analysis areas is then examined to identifywhether any target cells are present.

One advantage of the present invention is found in a blood separatingapparatus that can separate the entire buffy coat of a relatively largeblood sample from the rest of the blood volume.

Another advantage of the invention resides in the fact that the buffycoat layers can be made available for visualization or imaging in onesimple operation, i.e., the application of pressure and/orcentrifugation.

Still another advantage of the invention resides in enhanced buffy coatseparation, retention, and, if desired, removal from the sample tube forfurther processing.

Yet another advantage of the invention is found in that the toleranceprecision between the float and tube is decreased over that necessaryfor the prior art QBC-type systems, thus reducing the necessary cost ofthe components.

Still another advantage is found in that the tube can be supported forimproved imaging of the sample, and a more repeatable depth for imagingmay be provided.

Still further advantages of the present invention reside in itsrelatively simple construction, ease of manufacture, and low cost.

In a still additional aspect, the compressibility and/or rigidity of theflexible tube and rigid float can be reversed. In this aspect, the floatis designed to shrink in diameter at the higher pressures and movesfreely within a rigid, or optionally, semi-rigid tube. The use of acompressible float allows for usage of transparent glass tubes which, insome instances, exhibit enhanced optical properties over polymerictubes. Furthermore, this aspect generally reduces the tolerancerequirements for the glass tubes (since the float would expand upagainst the tube wall after the pressure decreases), and a full range offloat designs is possible.

In another aspect, the step of centrifugation is not required. In suchan aspect, the application of pressure alone to the inside of the tube,or simply the expansion of the tube (or the compression of the float),is required. For example, such pressure can be produced through the useof a vacuum source on the outside of the tube. Such an application alsoallows for the top of the sample tube to be kept open and easilyaccessible. Additionally, the use of a vacuum source may be easier toimplement in some situations than the application of a centrifugalforce.

Additionally, any method of tubular expansion/contraction (or floatcompression) such as mechanical, electrical, magnetic, etc., can beimplemented. Once the tube is expanded (or the float is compressed), thefloat will move to the proper location due to buoyancy forces created bythe density variations within the sample.

In a further aspect, the float comprises a part of a collection tubesystem or assembly. In this aspect, it is not necessary to transfer thesample from a collection container to an analysis tube. The blood orsample fluid can be collected immediately and then tested. Such a systemis somewhat faster, and also safer from a biohazard standpoint. Forexample, this system is desirable in very contagious situations (i.e.Ebola virus, HIV, etc.) where any type of exposure of the blood must beminimized.

Still further advantages and benefits of the present invention willbecome apparent to those of ordinary skill in the art upon reading andunderstanding the following detailed description of the preferredembodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may take form in various components and arrangements ofcomponents, and in various steps and arrangements of steps. Thedrawings, in which like reference numerals denote like componentsthroughout the several views, are only for purposes of illustratingvarious embodiments of the invention and are not to be construed aslimiting the invention.

FIG. 1 is a sectional view of a sample tube containing a generallyspool-shaped separator float according to an exemplary embodiment of theinvention.

FIG. 2 is an elevational view of a separator float having generallyconical ends according to another exemplary embodiment of the invention.

FIG. 3 is an elevational view of a separator float having generallyfrustoconical ends according to another exemplary embodiment of theinvention.

FIG. 4 is an elevational view of a separator float according to yetanother exemplary embodiment, wherein the ends are generally convex ordome shaped.

FIG. 5 is an elevational view of a separator float according to stillanother exemplary embodiment having sealing ridges offset from the ends.

FIGS. 6-8 are elevational views of ribbed separator floats according tofurther exemplary embodiments of the invention.

FIG. 9 is an elevational view of a separator float according to anotherexemplary embodiment of the present invention having generally helicaltube support ridges.

FIG. 10 is an elevational view of a separator float according to afurther embodiment of the invention having support ribs, which aretapered in the radial direction.

FIG. 11 is an elevational view of a separator float according to yetanother exemplary embodiment of the present invention having generallytapered, helical tube support ridges.

FIG. 12 is an elevational view of a separator float according to anotherembodiment of the invention having support ribs, which are rounded incross-sectional shape.

FIG. 13 is an elevational view of a separator float according to anotherembodiment of the invention having helical support ridges, which arerounded in cross-sectional shape.

FIG. 14 is an elevational view of a splined separator float according toanother exemplary embodiment of the invention.

FIG. 15 is an enlarged cross-sectional view taken along the lines 15-15shown in FIG. 14.

FIG. 16 is an elevational view of a further splined separator floatembodiment of the invention.

FIGS. 17 and 18 are elevational views of additional splined floatembodiments in accordance the invention.

FIG. 19 is a perspective view of yet another splined float embodiment ofthe present invention.

FIG. 20 is a perspective view of a float of still another exemplaryembodiment wherein the support ridges include intersecting annular ribsand splines.

FIGS. 21-26 are elevational views of knobbed or studded separator floatshaving generally rounded protrusions in various configurations, inaccordance with further exemplary embodiments of the present invention.

FIGS. 27 and 28 are elevational views of spiked or studded separatorfloats having facet-like protrusions according to additional exemplaryembodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Turning now to the drawings, wherein the showings are for purposes ofillustrating the preferred embodiments of the invention only and not forlimiting the same, FIG. 1 shows a blood separation tube and floatassembly 100, including a sample tube 130 having a separator float orbobber 110 of the invention therein.

The sample tube 130 is generally cylindrical in the depicted embodiment,although tubes having polygonal and other geometrical cross-sectionalshapes are also contemplated. The sample tube 130 includes a first,closed end 132 and a second, open end 134 receiving a stopper or cap140. Other closure means are also contemplated, such as parafilm or thelike. In alternative embodiments, not shown, the sample tube may be openat each end, with each end receiving an appropriate closure device.

Although the tube is depicted as generally cylindrical, the tube 130 maybe minimally tapered, slightly enlarging toward the open end 134,particularly when manufactured by an injection molding process. Thistaper or draft angle is generally necessary for ease of removal of thetube from the injection molding tool.

The tube 130 is formed of a transparent or semi-transparent material andthe sidewall 136 of the tube 130 is sufficiently flexible or deformablesuch that it expands in the radial direction during centrifugation,e.g., due to the resultant hydrostatic pressure of the sample undercentrifugal load. As the centrifugal force is removed, the tube sidewall136 substantially returns to its original size and shape.

The tube may be formed of any transparent or semi-transparent, flexiblematerial (organic and inorganic), such as polystyrene, polycarbonate,styrene-butadiene-styrene (“SBS”), styrene/butadiene copolymer (such as“K-Resin®” available from Phillips 66 Co., Bartlesville, Okla.), etc.Preferably, the tube material is transparent. However, the tube does notnecessarily have to be clear, as long as the receiving instrument thatis looking for the cells or items of interest in the sample specimen can“see” or detect those items in the tube. For example, items of very lowlevel of radioactivity that can't be detected in a bulk sample can bedetected through a non-clear or semi-transparent wall after it isseparated by the process of the present invention and trapped near thewall by the float 110 as described in more detail below.

In a preferred embodiment, the tube 130 is sized to accommodate thefloat 110 plus at least about five milliliters of blood or sample fluid,more preferably at least about eight milliliters of blood or fluid, andmost preferably at least about ten milliliters of blood or fluid. In anespecially preferred embodiment, the tube 130 has an inner diameter 138of about 1.5 cm and accommodates at least about ten milliliters of bloodin addition to the float 110.

The float 110 includes a main body portion 112 and two sealing rings orflanges 114, disposed at opposite axial ends of the float 110. The float110 is formed of one or more generally rigid organic or inorganicmaterials, preferably a rigid plastic material, such as polystyrene,acrylonitrile butadiene styrene (ABS) copolymers, aromaticpolycarbonates, aromatic polyesters, carboxymethylcellulose, ethylcellulose, ethylene vinyl acetate copolymers, nylon, polyacetals,polyacetates, polyacrylonitrile and other nitrile resins,polyacrylonitrile-vinyl chloride copolymer, polyamides, aromaticpolyamides (aramids), polyamide-imide, polyarylates, polyarylene oxides,polyarylene sulfides, polyarylsulfones, polybenzimidazole, polybutyleneterephthalate, polycarbonates, polyester, polyester imides, polyethersulfones, polyetherimides, polyetherketones, polyetheretherketones,polyethylene terephthalate, polyimides, polymethacrylate, polyolefins(e.g., polyethylene, polypropylene), polyallomers, polyoxadiazole,polyparaxylene, polyphenylene oxides (PPO), modified PPOs, polystyrene,polysulfone, fluorine containing polymer such aspolytetrafluoroethylene, polyurethane, polyvinyl acetate, polyvinylalcohol, polyvinyl halides such as polyvinyl chloride, polyvinylchloride-vinyl acetate copolymer, polyvinyl pyrrolidone, polyvinylidenechloride, specialty polymers, and so forth, and most preferablypolystyrene, polycarbonate, polypropylene, acrylonitritebutadiene-styrene copolymer (“ABS”) and others.

In this regard, one of the objectives of the present invention is toavoid the use of materials and/or additives that interfere with thedetection or scanning method. For example, if fluorescence is utilizedfor detection purposes, the material utilized to construct the float 110must not have interfering or “background” fluorescence at the wavelengthof interest.

The main body portion 112 and the sealing rings or support members 114of the float 110 are sized to have an outer diameter 118 which is lessthan the inner diameter 138 of the sample tube 130, under pressure orcentrifugation. The main body portion 112 of the float 110 is also lessthan the sealing or support rings 114, thereby defining an annularchannel 150 between the float 110 and the sidewall 136 of the tube 130.The main body portion occupies much of the cross-sectional area of thetube, the annular gap 150 being large enough to contain the cellularcomponents of the buffy coat layers and associated target cells when thetube is the non-flexed state. Preferably, the dimensions 118 and 138 aresuch that the annular gap 150 has a radial thickness ranging from about25-250 microns, most preferably about 50 microns.

While in some instances the outer diameter 118 of the main body portion112 of the float 110 may be less than the inner diameter 138 of the tube130, this relationship is not required. This is because once the tube130 is centrifuged (or pressurized), the tube 130 expands and the float110 moves freely. Once the centrifugation (or pressurization) step iscompleted, the tube 130 constricts back down on the sealing rings orsupport ridges 114. The annular gap or channel 150 is then created, andsized by the height of the support ridges or sealing rings 114 (i.e.,the depth of the “pool” is equal to the height of the support ridges114, independent of what the tube diameter is/was).

In an especially preferred embodiment, the float dimensions are 3.5 cmtall×1.5 cm in diameter, with a main body portion sized to provide a50-micron gap for capturing the buffy coat layers of the blood. Thus,the volume available for the capture of the buffy coat layer isapproximately 0.08 milliliter. Since the entire buffy coat layer isgenerally less than about 0.5% of the total blood sample, the preferredfloat accommodates the entire quantity of buffy layer separated in aneight to ten milliliter sample of blood.

The sealing or support flanged ends 114 are sized to be roughly equalto, or slightly greater than, the inner diameter 138 of the tube. Thefloat 110, being generally rigid, can also provide support to theflexible tube wall 136. Furthermore, the large diameter portions 114provide a sealing function to maintain separation of the bloodconstituent layers. The seal formed between the large diameter regions114 of the float and the wall 136 of the tube may form a fluid-tightseal. As used herein, the term “seal” is also intended to encompassnear-zero clearance or slight interference between the flanges 114 andthe tube wall 136 providing a substantial seal which is, in most cases,adequate for purposes of the invention.

The sealing rings 114 are most preferably continuous ridges, in whichcase the sample may be centrifuged at lower speeds and slumping of theseparated layers is inhibited. However, in alternative embodiments, thesealing ridges can be discontinuous or segmented bands having one oropenings providing a fluid path in and out of the annular gap 150. Thesealing ridges 114 may be separately formed and attached to the mainbody portion 112. Preferably, however, the sealing ridges 114 and themain body portion 112 form a unitary or integral structure.

The overall specific gravity of the separator float 110 should bebetween that of red blood cells (approximately 1.090) and that of plasma(approximately 1.028). In a preferred embodiment, the specific gravityis in the range of from about 1.089-1.029, more preferably from about1.070 to about 1.040, and most preferably about 1.05.

The float may be formed of multiple materials having different specificgravities, so long as the overall specific gravity of the float iswithin the desired range. The overall specific gravity of the float 110and the volume of the annular gap 150 may be selected so that some redcells and/or plasma may be retained within the annular gap, as well asthe buffy coat layers. Upon centrifuging, the float 110 occupies thesame axial position as the buffy coat layers and target cells and floatson the packed red cell layer. The buffy coat is retained in the narrowannular gap 150 between the float 110 and the inner wall 136 of the tube130. The expanded buffy coat region can then be examined, underillumination and magnification, to identify circulating epithelialcancer or tumor cells or other target analytes.

In one preferred embodiment, the density of the float 110 is selected tosettle in the granulocyte layer of the blood sample. The granulocytessettle on, or just above, the packed red-cell layer and have a specificgravity of about 1.08-1.09. In this preferred embodiment, the specificgravity of the float is in this range of from about 1.08 to about 1.09such that, upon centrifugation, the float settles in the granulocytelayer. The amount of granulocytes can vary from patient to patient by asmuch as a factor of about twenty. Therefore, selecting the float densitysuch that the float settles in the granulocyte layer is especiallyadvantageous since loss of any of the lymphocyte/monocyte layer, whichsettles just above the granulocyte layer, is avoided. Duringcentrifugation, as the granulocyte layer increases in size, the floatsettles higher in the granulocytes and keeps the lymphocytes andmonocytes at essentially the same position with respect to the float.

The method for detecting circulating epithelial cancer cells in a bloodof a subject is disclosed in U.S. Pat. No. 6,197,523 may advantageouslybe modified to employ the sample tube and float system of the subjectinvention. The aforementioned U.S. Pat. No. 6,197,523 is incorporatedherein by reference in its entirety.

In a preferred exemplary method of using the tube/float system 100 ofthe invention, a sample of anticoagulated blood is provided. Forexample, the blood to be analyzed may be drawn using a standardVacutainer® or other like blood collection device of a type having ananticoagulant predisposed therein.

A fluorescently labeled antibody, which is specific to the targetepithelial cells or other target analytes of interest, can be added tothe blood sample and incubated. In an exemplary embodiment, theepithelial cells are labeled with anti-epcam having a fluorescent tagattached to it. Anti-epcam binds to an epithelial cell-specific sitethat is not expected to be present in any other cell normally found inthe blood stream. A stain or colorant, such as acridine orange, may alsobe added to the sample to cause the various cell types to assumedifferential coloration for ease of discerning the buffy coat layersunder illumination and to highlight or clarify the morphology ofepithelial cells during examination of the sample.

The blood is then transferred to the assembly 100 for centrifugation.The float 110 may be fitted into the tube 130 after the blood sample isintroduced into the sample tube 130 or otherwise may be placed thereinbeforehand. The tube and float assembly 100 containing the sample isthen centrifuged. Operations required for centrifuging the blood bymeans of the subject tube/float system 100 are not expressly differentfrom the conventional case, although, as stated above, reducedcentrifuge speeds may be possible and problems of slumping may bereduced. An adaptor may optionally be utilized in the rotor to preventfailure of the flexible tube due to stress.

When the centrifugation is started, the resultant hydrostatic pressuredeforms or flexes the wall 136 so as to enlarge the diameter of thetube. The blood components and the float 110 are thus free to move undercentrifugal force within the tube 130. The blood sample is separatedinto six distinct layers according to density, which are, from bottom totop: packed red blood cells, reticulocytes, granulocytes,lymphocytes/monocytes, platelets, and plasma. The epithelial cellssought to be imaged tend to collect by density in the buffy coat layers,i.e., in the granulocyte, lymphocyte/monocyte, and platelet layers. Dueto the density of the float, it occupies the same axial position as thebuffy coat layers which thus occupy the narrow annular gap 150,potentially along with a small amount of the red cell and/or plasma).

After centrifugal separation is complete and the centrifugal force isremoved, the tube 130 returns to its original diameter to capture orretain the buffy coat layers and target analytes within the annular gap150. The tube/float system 100 is transferred to a microscope or opticalreader to identify any target analytes in the blood sample.

FIGS. 2-28 illustrate several exemplary modifications of the floataccording to the invention. FIG. 2 illustrates a float 210 that issimilar to the float 110 shown and described by way of reference to theof FIG. 1, which includes a main body portion 212 and sealing rings 214,but which further including a tapered or cone-shaped endcap member 216disposed at each end. The tapered endcaps 216 are provided to facilitateand direct the flow of cells past the float 210 and sealing ridges 214during centrifugation.

FIG. 3 illustrates a float 310, which is similar to the float 210 shownand described by way of reference to FIG. 2, including a main bodyportion 312 and sealing ridges 314, but having truncated cone-shapedendcap members 316, disposed at each end. The frustoconical endcaps 316are provided to facilitate the movement or flow of cells and the floatduring centrifugation.

FIG. 4 illustrates a float 410, which is substantially as shown anddescribed by way of reference to the floats 210 and 310 of FIGS. 2 and3, respectively, but where instead, generally convex or dome-shapedmembers 416, which cap the sealing ridges 414. The endcaps 416 may behemispherical, hemiellipsoidal, or otherwise similarly sloped, areprovided. Again, the sloping ends 416 are provided to facilitatedensity-motivated cell and float movement during centrifugation.

The geometrical configurations of the endcap units 216, 316, and 416illustrated in FIGS. 2-4, respectively, are intended to be exemplary andillustrative only, and many other geometrical shapes (including concaveor convex configurations) providing a curved, sloping, and/or taperedsurface around which the blood sample may flow during centrifugation.Additional exemplary shapes contemplated include, but are not limited totectiform and truncated tectiform; three, four, or more sided pyramidaland truncated pyramidal, ogival or truncated ogival; geodesic shapes,and the like.

FIG. 5 illustrates a float 510 similar to the embodiment depicted inFIG. 1, but wherein the sealing ridges are 514 are axially displacedfrom the ends. Optional endcap members 516 appear as conical in theillustrated embodiment. However, it will be recognized that the endcaps516, if present, any other geometrical configuration which provides asloped or tapered surface may be used, as described above.

Although the remaining FIGS. 6-28 are illustrated with generally flatends, i.e., without tapered ends, it will be recognized that each of theillustrated embodiments may optionally be modified to include any of theend cap types shown above in FIGS. 2-5, or other geometricalconfiguration which provide a sloped or tapered surface.

FIGS. 6-13 illustrate embodiments of the invention having generallyannular tube support members. FIG. 6 illustrates a ribbed float 610having a plurality of annular ribs or ridges 620 axially spaced along acentral body portion 612. Optional end sealing ridges 614 are disposedat opposite ends of the float. The ribs 620 and the optional end sealingridges 614 are sized to provide a sealing engagement with the tube 130(FIG. 1) when a centrifugal force is removed. The flexible tube expandsduring centrifugation to permit flow therearound during thedensity-based centrifugal separation process. The main body portion 612has a diameter smaller than the inner diameter of the tube duringcentrifugation and while supported by rib 614 and, thus, multipleannular channels 650 are defined between the main body portion 612 andthe inner tube wall upon completion of the centrifugation process.

Although the illustrated embodiment in FIG. 6 depicts continuous ribs,it will be recognized that the support ribs may likewise be broken orsegmented to provide an enhanced flow path between adjacent annularchannels 650. Additionally, multiple ribs and/or sealing ridges may bepresent in order to provide support for the deformable tube and/or toprevent the tube walls from collapsing inwardly.

FIG. 7 illustrates a float 710 according to a further embodiment. Thefloat 710 is similar to the float 610 shown in FIG. 6, and has aplurality of ribs 720 axially spaced along a central body portion 712,and wherein plural annular channels 750 are defined therebetween asdescribed above, but wherein the tube support ribs 720 are less denselyspaced apart than in the FIG. 6 embodiment. Optional sealing ridges 714are disposed at opposite ends of the float. Again, the illustratedembodiment depicts continuous ribs, however, it will be recognized thatthe support ribs may likewise be broken or segmented to provide anenhanced flow path between adjacent annular channels 750.

FIG. 8 illustrates a further float embodiment 810, similar to theembodiments of FIGS. 6 and 7, the above descriptions of which areequally applicable thereto. However, the float 810 differs in that itlacks sealing ridges at the opposite ends thereof, which may optionallybe provided, and the spacing of the ribs 820 is intermediate the ribspacing shown in FIGS. 6 and 7.

FIG. 9 illustrates a further float embodiment 910, wherein a helicalsupport member or ridge 920 is provided. That is, instead of discreteannular bands, multiple turns of the helical ridge 920 provides a seriesof spaced apart ridges on the main body portion 912, which defines acorresponding helical channel 950. The helical ridge 920 is illustratedas continuous, however, the helical band may instead be segmented orbroken into two or more segments, e.g., to provide path for fluid flowbetween adjacent turns of the helical buffy coat retention channel 950.Optional sealing ridges 914 appear at each axial end of the float 910.

FIGS. 10 and 11 illustrate further ribbed and helical float embodiments1010 and 1110, respectively. In FIG. 10, annular support ribs 1020, on amain body portion 1012, are tapered in the radial dimension. In FIG. 11,a tapered helical support 1120 appears, formed on a main body portion1112. The floats 1010 and 1110 are otherwise as described above by wayof reference to FIGS. 6 and 9, respectively. Although the supportmembers 1020 and 1120 are shown as continuous, they may alternatively bediscontinuous or segmented to facilitate axial flow. Option sealingridges, as described above, at opposite axial ends of the floats 1010and 1110 are omitted in the illustrated embodiment, and may optionallybe provided.

FIGS. 12 and 13 illustrate still further ribbed and helical floatembodiments 1210 and 1310, respectively. Appearing are support members1220 and 1320, formed on respective main body portions 1212 and 1312.The tube support members 1220 and 1320 each have a generally curved orrounded cross-sectional profile. The floats 1210 and 1310 are otherwiseas described above by way of reference to FIGS. 6 and 9, respectively.Again, the support members 1220 and 1320 are shown as continuous butmay, in alternative embodiments, be discontinuous or segmented. Optionalend sealing ridges 1314 appear in FIG. 13. Furthermore, end sealingridges do not appear in FIG. 12, but may optionally be provided.

Referring now to FIGS. 14 and 15, there is shown a splined separatorfloat 1410. The float 1410 includes a plurality of axially-orientedsplines or ridges 1424 radially spaced about a central body portion1412. Optional end sealing ridges 1414 are disposed at opposite ends ofthe float. The splines 1424 and the optional end sealing ridges 1414protrude from the main body 1412 to engage and provide support for thedeformable tube. Where provided, the end sealing ridges 1414 provide asealing function as described above. The axial protrusions 1424 definefluid retention channels 1450, between the tube inner wall and the mainbody portion 1412. The surfaces 1413 of the main body portion disposedbetween the protrusions 1424 may be curved, e.g., when the main bodyportion is cylindrical, however, flat surfaces 1413 are alsocontemplated. Although the illustrated embodiment depicts splines 1424that are continuous along the entire axial length of the float,segmented or discontinuous splines are also contemplated.

FIG. 16 illustrates a further splined float embodiment 1610 similar tothe float 1410 as shown and described above by way of reference to FIGS.14 and 15, but wherein optional end sealing ridges are not provided.

FIGS. 17 and 18 are elevational views of alternative splined floats 1710and 1810, respectively, and are similar to the respective embodimentsshown and described above by way of reference to respective FIGS. 14 and16, but wherein the axial splines 1724 and 1824, respectively,protruding from respective main body portions 1712 and 1812 are moresparsely radially spaced. The float 1710 includes optional end sealingridges 1714; such do not appear on the float 1810 of FIG. 18. As above,the respective surfaces 1713 and 1813 may be flat or curved.

Referring now to FIG. 19, there is shown a perspective view of a splinedseparator float 1910 in accordance with a further embodiment of theinvention. Multiple axially oriented splines 1924 are spaced radiallyabout and protrude from a central body portion 1912 to provide supportfor the flexible tube. Optional sealing end ridges 1914 are disposed atopposite ends of the float 1910. Fluid retention channels 1950 formedbetween adjacent splines 1924 are defined by adjacent splines 1924 andsurfaces 1913 on the main body portion 1912. The surfaces 1913 aredepicted as generally flat, although curved surfaces are alsocontemplated. The axial splines 1924 are depicted as continuous alongthe length of the tube; however, segmented or discontinuous splines arealso contemplated.

Referring now to FIG. 20, there is shown yet another embodiment 2010,including a tube supporting member 2026 protruding with respect to amain body portion 2012. The support means 2026 can be described as anintersecting network of annular rings or ribs 2020 and axial splines2024. Optional end sealing ridges 2014 are disposed at opposite ends ofthe float. The support member 2026 and the optional sealing ridges 2014radially protrude from the main body portion 2012 at opposite ends ofthe float to engage and provide support for the deformable tube. Whereprovided, the end sealing ridges 2014 provide a sealing function asdescribed above. The raised support member 2026 defines a plurality offluid retention windows 2050 formed between the tube inner wall and themain body portion 2012. Surfaces 2013 of the main body portion 2012corresponding to the windows 2050 may be curved, e.g., when the mainbody portion is cylindrical, however, flat surfaces 2013 are alsocontemplated. Although the illustrated embodiment depicts the supportmember 2026 as a network of annular ribs and axial splines which iscontinuous, breaks may also be includes in the annular and/or axialportions of the network 2026, e.g., to provide a fluid path between twoor more of the windows 2050.

FIGS. 21-26 illustrate several floats having a plurality of protrusionsthereon for providing support for the deformable walls of the sampletube. Referring to FIGS. 21 and 22, float 2110 and 2210, respectively,include multiple rounded bumps or knobs 2128 spaced over the surface ofa central body portion 2112. Optional end sealing ridges 2114 (FIG. 21)are disposed at opposite ends of the float 2110 and do not appear on thefloat 2210 of FIG. 22. The knobs 2128 and the optional end sealingridges 2114 radially protrude from the main body 2112 and traverse anannular gap 2150 to engage and provide support for the deformable tubewall. Where provided, the end sealing ridges 2114 provide a sealingfunction as described above. The surface of the main body portiondisposed between the protrusions may be curved, e.g., when the main bodyportion is cylindrical, or, alternatively, may have flat portions orfacets.

In FIGS. 23 and 24, there are illustrated float embodiments 2310 and2410, which are as substantially as described above by way of referenceto FIGS. 21 and 22, respectfully, but wherein the protrusions 2328 forman aligned rather than staggered pattern over the surface of the mainbody portion 2312. Optional end sealing ridges 2314 appear in the FIG.23 embodiment.

Referring now to FIGS. 25 and 26, there are illustrated floatembodiments 2510 and 2610, which are as substantially as described aboveby way of reference to FIGS. 21 and 22, respectfully, but wherein theprotrusions 2528 are less densely spaced over the surface of the mainbody portion 2512. Optional end sealing ridges 2514 appear in the FIG.25 embodiment.

FIGS. 27 and 28 illustrate float embodiments 2710 and 2810,respectively, which include multiple raised facets 2728 spaced over thesurface of a central body portion 2712. Optional end sealing ridges 2714(FIG. 27) are disposed at opposite ends of the float 2710, and do notappear in the FIG. 28 embodiment. The facets 2728 and the optional endsealing ridges 2714 radially protrude from the main body 2712 andtraverse an annular gap to engage and provide support for the deformabletube wall and define a plurality of fluid retention windows 2750. Whereprovided, the end sealing ridges 2714 provide a sealing function asdescribed above. The surfaces 2713 of the main body portion, disposedbetween the protrusions 2728 and forming a surface defining thefluid-retention windows 2750, may be curved surfaces, e.g., when themain body portion is cylindrical. Alternatively, the surfaces 2713 maybe flat. In alternative embodiments, the size, spacing density, andalignment patterns of the facets 2718 can be modified extensively.

The exemplary embodiments of FIGS. 21-28 have been described withreference to rounded knobs or square facets as supporting the flexiblesample tube, although protrusions of any geometrical configuration maybe used. Other geometrical configurations for the protrusions are alsocontemplated, such as conical or frustoconical spikes, tectiform ortruncated tectiform protrusions, cylindrical protrusions, pyramidal ortruncated pyramidal protrusions, hemiellipsoidal protrusions, and soforth, as well as any combinations thereof. Likewise, the size, spacing,and pattern of the protrusions can be varied. Where the sample is to beimaged, the size and spacing can be selected in accordance with theimaging field of view and other factors.

The invention has been described with reference to the preferredembodiments. Obviously, modifications and alterations will occur toothers upon reading and understanding the preceding detaileddescription. It is intended that the invention be construed as includingall such modifications and alterations insofar as they come within thescope of the appended claims or the equivalents thereof.

1. A volume occupying separator float to be inserted into an associatedsample tube, comprising: a main body portion and one or more supportmembers protruding from the main body portion to engage and support asidewall of the sample tube, said main body portion and said supportmembers having a cross-sectional diameter less than a first innerdiameter of the tube when the sample tube is expanded, wherein said mainbody portion together with an axially aligned portion of said sidewalldefine an annular gap therebetween; wherein said support memberstraverse said annular gap to produce one or more analysis areas.
 2. Theseparator float according to claim 1, wherein the main body portion andthe one or more support members are integrally formed.
 3. The separatorfloat according to claim 1, wherein the annular gap has a radialthickness of about 50 microns.
 4. The separator float according to claim1, wherein the sample tube is sized to receive a blood sample ofapproximately ten milliliters in volume.
 5. The separator floataccording to claim 4, wherein the annular gap has a volume of about 0.08milliliter.
 6. The separator float according to claim 1, wherein thefloat includes opposite axial ends which are tapered in the axialdirection.
 7. The separator float according to claim 1, wherein the oneor more support members include one or more annular ridges.
 8. Theseparator float according to claim 1, wherein the one or more supportmembers include two annular ridges.
 9. The separator float according toclaim 8, wherein the two annular ridges are disposed at opposite axialends of the float.
 10. The separator float according to claim 1, whereinthe one or more support members include three or more axially-spacedannular ridges.
 11. The separator float according to claim 1, whereinthe one or more support members comprises a helical ridge.
 12. Theseparator float according to claim 1, wherein the one or more supportmembers include a plurality of circumferentially spaced-apart splines.13. The separator float according to claim 12, wherein the splines arealigned parallel to an axis of the float.
 14. The separator floataccording to claim 1, wherein the one or more support members furtherinclude annular ridges disposed at opposite axial ends of the float. 15.The separator float according to claim 1, wherein the one or moresupport members include a plurality of radially spaced-apart splinesintersecting with a plurality of axially spaced-apart splines.
 16. Theseparator float according to claim 1, wherein the one or more supportmembers include a plurality of raised protrusions spaced over thesurface of the main body portion.
 17. The separator float according toclaim 16, wherein the protrusions are selected from rounded bumps andfaceted bumps.
 18. The separator float according to claim 1, wherein theannular gap has a radial thickness of from about 25 to about 250microns.
 19. The separator float according to claim 1, wherein, when thesample tube receives a blood sample, the annular gap has a volume largeenough to contain from about 1 to about 100 rare cells per milliliter ofthe blood sample.